Construction and application of double-block DNA-based regulable nanogold probe

A nano-gold probe and double-stranded probe technology, applied in the biological field, to achieve the effect of solving non-specific adsorption

Pending Publication Date: 2019-10-01
SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, despite their individual efficiency, sensitivity, and convenience, none of the methods have tunable sensitivity and signal

Method used

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  • Construction and application of double-block DNA-based regulable nanogold probe
  • Construction and application of double-block DNA-based regulable nanogold probe
  • Construction and application of double-block DNA-based regulable nanogold probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] (1) Materials and equipment

[0050] Reagents used in this embodiment: trisodium citrate (C6H5Na3O7.2H2O), phosphate, NaCl, MgCl2 and KCl and other reagents were purchased from Sinopharm.

[0051] The above reagents were of analytical grade without further purification.

[0052] The water used in this embodiment is MilliQ water; MilliQ water: 18.2 MΩ.cm (Millipore). The equipment used in this example includes a transmission electron microscope (TEM), an ultraviolet spectrophotometer (Hitachi U-3010), a fluorescence spectrophotometer (F-900, Edinburg), a pH meter, and a refrigerated high-speed centrifuge (Hitachi).

[0053] The diblock DNA, reporter molecule and target molecule used in the embodiment of the present invention were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. and purified by high performance liquid chromatography.

[0054] The embodiment of the present invention uses three diblock DNAs, namely 2blockPolyA30 (SEQ ID NO.1), 2blockPolyA40 (SEQ ...

Embodiment 2

[0067] In this example, the regulation of the number of assembled gold nanoparticles on the surface of the diblock DNA with different Poly A lengths is discussed.

[0068] The diblock DNA nano-gold probe solutions with different lengths of Poly A prepared in Example 1 were first quantified by an ultraviolet spectrophotometer. The calculation formula is CAuNPs-PolyA=A520nm*2.79*10-10M. The diblock nano-gold probe (0.15 nM final concentration) was added to mercaptoethanol (20 mM final concentration) and shaken overnight at room temperature. Then the supernatant fluorescently labeled DNA (fluorophore FAM, excitation wavelength 494nm, emission wavelength 520nm) was collected by centrifugation, and the fluorescence intensity was detected. Finally, the fluorescence value was substituted into the previous standard curve to calculate the concentration of fluorescently labeled DNA. like Figure 4 As shown, (A) As the length of PolyA increases, the number of diblock DNA assembled on ...

Embodiment 3

[0070] In this example, the regulation of the recognition ability of the target sequence by diblock DNA with different numbers of Poly A nucleotides is discussed.

[0071] Nanogold probes with different lengths of Poly A provided in Example 1 were used. Add different concentrations of target molecules to different groups of nano-gold probes, respectively: 0, 0.01, 0.05, 0.1, 0.5, 1, 10, 50, 100, 150, 200nM, and detect the fluorescence intensity of each group after incubation. Each experiment was repeated three times. In this embodiment, the sequence of the target molecule is shown in SEQ ID NO.11, specifically 5' AGCCC CTGCC CACCG CACAC TG3'.

[0072] The result is as Figure 5 As shown, the intensity of the fluorescent signal increases with the addition of target molecules, and finally reaches a plateau. Different poly A numbers have a regulatory effect on the minimum detection limit of the probe. As the number of adenine nucleotides in PolyA increases, the detection limi...

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Abstract

The invention relates to the technical field of biology, and in particular relates to a nanogold probe. The nanogold probe comprises nanogold particles and a double-chain probe connected to the nanogold particles, wherein the double-chain probe comprises a first chain and a second chain; the first chain comprises a first sector and a second sector which are arranged in sequence in a 5'-3' mode; the first sector and the second chain are complementary; the second sector is used as a sticky tail end and is used for recognizing a target sequence; and the second chain is modified by a fluorescent group. According to the nanogold probe provided by the invention, the nucleotide number of the second sector is adjusted, so that the hybridization efficiency of an identification sequence and the target sequence is regulated and controlled, and then the identification capability of the nanogold probe on the target sequence is regulated and controlled.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a double-block DNA-based adjustable nano-gold probe and its preparation and application. Background technique [0002] The detection of DNA / RNA has broad application space in biomedical research and clinical diagnosis. DNA detection is of great significance in molecular diagnosis of diseases, gene therapy, biomedical research, rapid detection of biological warfare agents and forensic applications. RNA plays an important role in biological processes, affecting the development of cells, tissues and individuals, and disease processes lead to specific RNAs being elevated in body fluids. For example, tumor-specific mRNA / miRNA can be used in the diagnosis and typing of cancer and has attracted extensive attention. [0003] Traditional nucleotide detection methods are divided into two categories, one is based on probe hybridization technology, including Northern blotting, microarray chip ...

Claims

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Application Information

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IPC IPC(8): G01N21/64C09K11/06
CPCG01N21/6428G01N21/6486C09K11/06G01N2021/6432
Inventor 宓现强王璐张欢
Owner SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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